Search

PIFE was first used as an acronym for protein-induced fluorescence enhancement, which refers to the increase in fluorescence observed upon the interaction of a fluorophore, such as a cyanine, with a protein. This fluorescence enhancement is due to changes in the rate of cis/trans photoisomerisation. It is clear now that this mechanism is generally applicable to interactions with any biomolecule and, in this review, we propose that PIFE is thereby renamed according to its fundamental working principle as photoisomerisation-related fluorescence enhancement, keeping the PIFE acronym intact. We discuss the photochemistry of cyanine fluorophores, the mechanism of PIFE, its advantages and limitations, and recent approaches to turn PIFE into a quantitative assay. We provide an overview of its current applications to different biomolecules and discuss potential future uses, including the study of protein-protein interactions, protein-ligand interactions and conformational changes in biomolecules., Comment: No Comments

Protein folding is an indispensable process for the majority of proteins after their synthesis from ribosomes in the cell. Most in vitro protein folding studies have focused on single-domain proteins. Hence, it is important to understand the folding process of multi-domain proteins, especially when domains are discontinuous. We choose the Maltose binding protein (MBP) as a model system. In particular, we studied a mutant of MBP that folds slowly. Here, using two- and three-color single-molecule Foerster resonance energy transfer (smFRET) experiments, we study the refolding of both the domains and the interaction between the domains of DM-MBP. Initial two-color smFRET measurements of the N-terminal domain (NTD) reveal the presence of a folding intermediate. The same folding intermediate is observed in measurements monitoring the C- terminal domain (CTD) and the NTD-CTD (N-C) interface. The refolding intermediate is dynamic on the sub-millisecond timescale. Quantitative analysis on underlying dynamic interconversions revealed a delay in NTD folding imposed by the entropic barrier being the primary cause for slow DM-MBP folding. Moreover, CTD folds after NTD completes the folding. Using three-color smFRET, we could show the NTD folds first and CTD later in a same protein. Molecular dynamic simulations for temperature-induced unfolding on WT- and DM-MBP identify a folding nucleus in the NTD, which is rich in hydrophobic residues, and explains why the two mutations slow down the folding kinetics. In the presence of the bacterial Hsp60 chaperonin system GroEL/ES, we observe that DM-MBP is dynamic within the chaperonin cavity but the chaperonin limits the conformational space of substrate. Hence, confinement aids DM-MBP in overcoming the entropic barrier. The study reports on the subtle tuning and co-dependency for protein folding between two-domains with a discontinuous arrangement.

By circumventing the optical diffraction limit, super-resolved fluorescence microscopies enable the study of larger cellular structures and molecular assemblies. However, fluorescence nanoscopy currently lacks the spatiotemporal resolution to resolve distances on the size of individual molecules and reveal the conformational fine structure and dynamics of molecular complexes. Here we establish FRET nanoscopy by combining colocalization STED microscopy with multiparameter FRET spectroscopy. We simultaneously localize donor and acceptor dyes of single FRET pairs with nanometer resolution and quantitatively measure intramolecular distances with sub-nanometer precision over a large dynamic range. While FRET provides isotropic 3D distance information, colocalization measures the projected distance onto the image plane. The combined information allows us to directly determine its 3D orientation using Pythagoras's theorem. Studying two DNA model systems and the human guanylate binding protein hGBP1, we demonstrate that FRET nanoscopy unravels the interplay between their spatial organization and local molecular conformation in a complex environment., Comment: Main Text (34 pages, 6 figures) with Supporting Information (90 pages, 29 figures, 20 tables)

Single-molecule F\"orster Resonance Energy Transfer (smFRET) is ideally suited to resolve the dynamics of biomolecules. A significant challenge to date is capturing and quantifying the exchange between multiple conformational states, mainly when these dynamics occur on the sub-millisecond timescale. Many methods for the quantitative analysis are challenged if more than two states are involved, and the appropriate choice of the number of states in the kinetic network is difficult. An additional complication arises if dynamically active molecules coexist with solely static molecules in similar conformational states. To address these problems, we developed an integrative analysis framework that combines the information from FRET-lines, time-correlated single photon counting, and fluorescence correlation spectroscopy. While individually, these methodologies provide ambiguous results for quantitatively characterize the dynamics in complex kinetic networks, the global analysis enables accurate determination of the number of states, their kinetic connectivity, and the transition rate constants. To challenge the potential of smFRET to study multi-state kinetic networks, we benchmark the framework using synthetic data of three-state systems with different kinetic connectivity and interconversion rates., Comment: Main Text (31 pages, 9 figures, 3 tables) with Supporting Information (40 pages, 10 figures, 2 tables)

Conformational dynamics of biomolecules are of fundamental importance for their function. Single-molecule F\"orster Resonance Energy Transfer (smFRET) is a powerful approach to inform on the structure and the dynamics of labeled molecules. If the dynamics occur on the sub-millisecond timescale, capturing and quantifying conformational dynamics can be challenging by intensity-based smFRET. Multiparameter fluorescence detection (MFD) addresses this challenge by simultaneously registering intensities and fluorescence lifetimes. Together, the mean donor fluorescence lifetime and the fluorescence intensities inform on the variance, and the mean FRET efficiency tells the conformational dynamics. Here, we present a general framework that relates average fluorescence lifetimes and intensities in smFRET counting histograms. Using this framework, we show how to compute parametric relations (FRET-lines) of these observables that facilitate a graphical interpretation of experimental data, can be used to test models, identify conformational states, resolve the connectivity of states, and can be applied to unstructured systems to infer properties of polymer chains or study fast protein folding. To simplify the graphical analysis of complex kinetic networks, we derive a moment-based representation of the experimental data and show how to decouple the motion of the fluorescence labels from the conformational dynamics of the biomolecule., Comment: Main Text (47 pages, 14 figures, 3 tables) with Supporting Information (22 pages, 2 figures)

Single-molecule F\"{o}rster resonance energy transfer (smFRET) has become a mainstream technique for probing biomolecular structural dynamics. The rapid and wide adoption of the technique by an ever-increasing number of groups has generated many improvements and variations in the technique itself, in methods for sample preparation and characterization, in analysis of the data from such experiments, and in analysis codes and algorithms. Recently, several labs that employ smFRET have joined forces to try to bring the smFRET community together in adopting a consensus on how to perform experiments and analyze results for achieving quantitative structural information. These recent efforts include multi-lab blind-tests to assess the accuracy and precision of smFRET between different labs using different procedures, the formal assembly of the FRET community and development of smFRET procedures to be considered for entries in the wwPDB. Here we delve into the different approaches and viewpoints in the field. This position paper describes the current "state-of-the field", points to unresolved methodological issues for quantitative structural studies, provides a set of 'soft recommendations' about which an emerging consensus exists, and a list of resources that are openly available. To make further progress, we strongly encourage 'open science' practices. We hope that this position paper will provide a roadmap for newcomers to the field, as well as a reference for seasoned practitioners.

Single-molecule F\"orster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards ensuring both the reproducibility and accuracy of measuring FRET efficiencies are currently lacking. Here we report the results of a worldwide, comparative, blind study, in which 20 labs determined the FRET efficiencies of several dye-labeled DNA duplexes. Using a unified and straightforward method, we show that FRET efficiencies can be obtained with a standard deviation between ${\Delta}$E = +-0.02 and +-0.05. We further suggest an experimental and computational procedure for converting FRET efficiencies into accurate distances. We discuss potential uncertainties in the experiment and the modelling. Our extensive quantitative assessment of intensity-based smFRET measurements and correction procedures serve as an essential step towards validation of distance networks with the ultimate aim to archive reliable structural models of biomolecular systems obtained by smFRET-based hybrid methods., Comment: 27 pages, 5 figures, supplementary material, corrections: p20 last sentence; eqn. 13 & 14